Inorganic phosphor labelled macromolecules; a process for their preparation and their use for immunological or immunocytochemical assays

ABSTRACT

The invention relates to macromolecules, such as proteins, including immunologically specific antibodies, lipoproteins, polynucleotides, etc., provided with luminescent labels. 
     The invention further relates to a process for preparing macromolecules, such as proteins, including immunologically specific antibodies, lipoproteins, polynucleotides, etc., provided with luminescent labels, and also to the use of labelled immunologically specific macromolecules, such as antibodies, for immunological or immunocytochemical assays.

The invention relates to macromolecules, such as proteins, includingimmunologically specific antibodies, lipoproteins, polynucleotides,etc., provided with luminescent labels.

The invention further relates to a process for preparing macromolecules,such as proteins, including immunologically specific antibodies,lipoproteins, polynucleotides, etc., provided with luminescent labels,and also to the use of labelled immunologically specific macromolecules,such as antibodies, for immunological or immunocytochemical assays.

In biomedical examinations, use is often made of the specificimmunological reaction between antigen and (monoclonal) antibody. Torender visible and quantify this reaction, the antigen or the antibodyis provided with a label. The most common labels are enzymes that can bedemonstrated with specific colour reactions, luminescent compounds, andradioactive isotopes. Prominent among luminescent labels arephotoluminescent compounds, such as fluorochromes, although thesuitability of bioluminescents and chemiluminescents has beendemonstrated (Pratt et al., 1978; Simpson et al., 1979; Hastings andWilson, 1976).

Apparatus for the qualitative and/or quantitative processing offluorescence signals (photons) has been greatly improved in the last fewyears. Single-photon-counting detection systems and highly sensitivecameras of the SIT or ISIT type (with image intensifiers), as well ashigh-energetic excitation sources, such as lasers, are at presentavailable. To increase the amount of fluorescence, new fluorochromes,such as phycobiliproteins, with a high fluorescence efficiency have beendeveloped (Oi et al., 1983). Polystyrene or latex micropheres, whichcontain thousands of fluorochrome molecules, have also been introducedas cell labels (so-called Covaspheres) (Molday et al., 1975; EuropeanPatent Application EP 0002963 (Eastman Kodak Company), 1979). Thanks tothese improvements, the extreme sensitivity of fluorescence studies ofcells and tissues is at persent determined for the major part by theautofluorescence of the biological object and the optics used (lensesand filters), and by undesirable excitation light from the radiationsource. The autofluorescence of a single cell, measured with amicroscope fluorimeter or a flow cytometer, is in the order of severalthousands of fluorescein equivalents (Jongkind et al., 1979), as aresult of which the demonstration of minute amounts of macromolecule incells and tissues by means of immunofluorescence is not optimallypossible. It is especially for the demonstration of minute amounts ofmacromolecules, therefore that one would often opt for a method using aradioactive label. This method is sensitive, it is true, but in the caseof in situ applications (e.g. autoradiography) extremely time-consuming,while in addition the necessary precautionary measures must be takenwith regard to working conditions and the processing of the radioactivewaste.

In analytical chemistry, the problem of autofluorescence can be largelyavoided by using so-called time-resolved fluorescence assays. For thismethod a luminescent label must be selected with a long after-glow time,such as the lanthanides europium (eu) and terbium (Tb). After a shortexciting light pulse, the relatively fast autofluorescence (in the orderof nanoseconds or faster) can be separated in time from the slowerlanthanide fluorescence (in the order of milliseconds). The DELFIA(Delayed Fluoro Immuno Assay) system, developed by LKB, makes use ofthis principle. This method has been found to be competitive insensitivity with radioactive assays (Soini et al., 1983; Hemmila et al.,1984). One disadvantage is, however, that the luminescent label Eu or Tbwhen bonded to antibodies and dissolved in water does not luminesce, orhardly so, and therefore after the reaction with the antigen-antibodymust be liberated to obtain the delayed-fluorescence properties. As aconsequence, detection is only possible in solutions, and uses of thismethod in which localisation of the antigen at the tissue, cell orchromosomal level is required are impossible. The very low quantumefficiency of the lanthanides in aqueous solutions can be improvedconsiderably by incorporating lanthanides such as e.g. Europium andTerbium by means of crosslinking agents into latex particles, therebyincreasing its fluorescence yield (EP 0002963; Eastman Kodak Company,1979). Thus prepared latex particles can be coupled to cells. It isthese very cellular uses which command increasing interest, thanks tothe development of molecular biology and the possibility ofdemonstrating oncogene and virus products in tissues and cells usingspecific monoclonal antibodies. The present inventors have developed anew immunological label which combines many of the advantages of theexisting methods, and in addition permits localisation of theantigen-antibody complex at cell level.

The invention is characterized in that the labels are inorganiccrystalline phosphors.

For use in biological systems, the size of the crystalline phosphorsmust generally not exceed 5 um. Preferably the labels used arecrystalline phosphors with a particle size of 1 um or smaller.

Because, on the other hand, the luminescence intensity decreases rapidlywith decreasing size of the phosphors, the phosphors should generallyhave a size of 0.02 um or larger. Preferably, the labels used arecrystalline phosphors with a particle size of 0.1 um or larger.

The invention accordingly provides a material comprising a label whichis a crystalline phosphor, to which the macromolecule, such as antibody,is adsorbed or chemically bonded. These phosphor labels luminesce blue,green or red (depending on the phosphor used) under a fluorescencemicroscope (UV excitation) or exhibit cathode luminescence in a scanningelectron microscope. As their after-glow time is in the order of msec orsec, phosphor-labelled cells can be measured in accordance with thetime-resolved principle with microscope-cyto fluorimeters and flowcytometers adapted for the purpose. With regard to the nature of theinorganic crystalline phosphors, there are no limitations. All knowninorganic crystalline phosphors, such as those described in Kirk-OthmerEncyclopedia of Chemical Technology, third edition, Volume 14, pp. 527ff., and in many patent publications, and also as yet unknown inorganiccrystalline phosphors are suitable for use as labels in accordance withthe present invention, with their particle size being preferably reducedto 1 um or less.

The use of a stable suspension of inorganic crystalline phosphors withmacromolecules (antibodies) physically or chemically attached, has notbeen described before as (immuno)cytochemical reagent. K. Kosak (U.S.Pat. No. 4,000,252; 1976) has mentioned the use of a phosphor-antibodycomplex in a new type of immunoscintillation cell for a newradioimmunoassay method. This cell consists of a support medium withorganic phosphors incorporated, to which antibodies are coupled. Thecell may have different physical shapes. The support medium serves as atrapping agent for the radioactively labelled or unlabelled antigen;binding of a radioactively labelled antigen induces luminescence of thephosphor in its direct vicinity, which is subsequently measured as isdone in a scintillation counter. The present patent application does notrelate to a solid phase consisting of an organic phosphor-antibodycomplex, but relates to a stable suspension of an inorganicphosphor-antibody complex, which is used as (immuno)cytochemicalreagent. In 1972 L. Amante et al. have studied the properties ofconjugates of immunoglobulins with the amorphous and crystalline form oftetramethyl rhodamine isothiocyanate (TRITC). However, the fluorophoreTRITC was dissolved from its various native states in water, andsubsequently coupled to immunoglobulin.

Before the coupling between the inorganic crystalline phosphors and themacromolecules, such as antibodies, is brought about, the phosphors,which as stated before preferably have particle sizes of 1 um or less,should preferably be subjected to a pre-treatment to provide theirsurface with charged groups. As a consequence the individual particleswill repel each other and the formation of larger aggregates will beavoided. The nature of the charged groups is not critical. Both groupsof negative charge, such as carboxylic acid groups, sulphonic acidgroups, phosphoric acid groups, and the like, and positively chargedgroups, such as amino groups and quaternary ammonium groups, aresuitable for the effect contemplated.

Experiments conducted and the results achieved therein (see the example)show that, in principle, it is possible to couple antibodies tocrystalline phosphors with a particle size of 1 um and smaller in such amanner that specific immunological reactions with this antibody-phosphorconjugate are possible. Of two tested methods, the adsorption ofantibodies to phosphors at a pH in the vicinity of the isoelectric point(for IgG 6.6) turned out to be preferred to covalent coupling viasuitable spacer molecules. The adsorption method is known per se and,among other applications, is successfully used for coating colloidalgold spheres with antibodies, for use in the electron microscope (Geuzeet al., 1981). The use of other coupling methods than the methods hereinillustrated, however, is not excluded.

Immunologically positive lymphocytes are easily recognized by means offluorescence microscopy with UV excitation. A major advantage is thatthe phosphors used do not exhibit bleaching. Crystals that have notreacted are easily distinguished as small phosphorescent dots in thebackground. Nevertheless, we prefer to separate the non-reacted phosphorcrystals from the cells, for example, by gradient centrifugation, as aresult of which a cleaner background is obtained. The relatively smallcrystals (about 0.2 um) can easily be removed by centrifugation, becausethese remain behind in the supernatant at (200-400)xg (conventional forcells). Tests in which a density gradient was used also gave goodresults. The luminescence intensity of phosphor-labelled cells was high.Microscope photography required exposure periods of 1 to 5 sec.Comparable conventional fluorescence colourings of membrane componentscommonly result in exposure times of 30 to 60 sec., partly owing to theoccurrence of bleaching.

The phosphors cannot only be rendered visible to the human eye by meansof the fluorescence microscope and the electron microscope. Measurementsof the luminescence signal and reproduction thereof by means of a memoryoscilloscope show that the time-resolved luminescence measuringprinciple can be used. This offers the possibility of adapting theapparatus currently available for the quantitative fluorescence analysisof tissues, cells and cell components in such a manner that the specific(slow) phosphorenscence signals can be separated from the interfering(fast) background fluorescence signals and from possibly reflectedexcitation light. An additional advantage is that the backgroundluminescence of biological objects such as cells virtually exclusivelyconcerns "fast" fluorescence processes.

The apparatus used for the quantitative analysis of cells mainlyconcerns microscope fluorimeters and flow cytometers. The detectionsystems of these fluorimeters mostly consist of photomultiplier tubes,photodiodes, television cameras, or arrays of detectors. With arelatively simple electronic circuit, a time delay can be realizedbetween the moment of excitation (in pulsed form) and the moment whenthe (delayed) luminescence signal is measured. This principle can beused in systems in which the entire microscopic object is bodilyexcited, but also in systems where the object is scanned point by pointby a small excitation light spot (laser scanning, incremental tablescanners).

In flow systems in which the excitation of cells flowing past and themeasurement of luminescence take place along different optical paths,the time delay can also be realized by positioning the lens registeringthe luminescence signal somewhat downstream. For example, in a FACS typecell sorter a delay of 100 microseconds can be realized by focussing thedetection lens 1 cm lower on the liquid stream.

USES OF PHOSPHOR-LABELLED IMMUNOLOGICAL REAGENTS

In principle, phosphor conjugates are suitable for use in allapplications where fluorochrome, enzyme, or isotope-labelledimmuno-reagents are used. Examples are ELISA and RIA techniques fordemonstrating and assaying antigens in solution, immunological methodsfor the detection of macromolecules in filter blots, andimmunocytochemical methods for the study of morphologically intacttissues and cells. As regards the cytochemical application the accentwill be on demonstrating superficial antigens, as phosphor particles of0.1-1.0 um cannot easily penetrate cell membranes.

By using phosphors, several parameters can be studied and measured atthe same time. Not only is it possible to generate three spectrallyseparate colours (blue, green, red) by means of UV or electronexcitation, but phosphors with different decay times can be used, byvirtue of which the number of antigens to be measured at the same timecan become very large.

Time-resolved luminescence assays are comparable in sensitivity toradioactivity assays (Soini and Kojola, 1983). The immunocytochemicaluse of phosphor conjugates basically allows a much more sensitivedetection of small quantities of macromolecules in cells. This may be ofimportance in both fundamental and diagnostic examination formembrane-linked oncogene proteins, viral products and differentiationantigens.

In summary, it has accordingly been found that macromolecules, such asimmunoglobulins can be adsorbed onto crystalline phosphors with aparticle size of 1 um or smaller, and that the resulting conjugates,such as phosphor-antibody conjugates, are immunologically andimmunocytochemically specific and applicable. Examples of the propertiesof the phosphors, other than their high physico-chemical stability arethat they can be rendered visible by excitation with UV light or with anelectron beam, and that the luminescence of the conjugates, such asphosphor-antibody conjugates, does not decrease during excitation (nobleaching). In addition, the luminescence belongs to the relatively slowluminescence (phosphorescence). As the luminescence decay is in theorder of milliseconds, a strong suppression of autofluorescence andundesirable background reflection is possible by means of time-resolvedluminescence assays, by virtue of which a high sensitivity can beachieved. The luminescence of phosphors can be observed with microscopefluorimeters and flow cytometers. These can be modified fortime-resolved luminescence assays in a relatively simple manner. The useof phosphor-antibody conjugates basically makes it possible to assay aplurality of antigens simultaneously, because the luminescence ofphosphors is not only well separated spectrally (blue, green, red), butalso exhibits measurable differences in decay times.

The invention is illustrated in and by the following example.

EXAMPLE (a) Preparation of the phosphors

The starting products were two types of phosphors: a blue one consistingof zinc sulfide (ZnS) activated with silver (Ag), and a red oneconsisting of yttrium oxysulfide (Y₂ O₂ S) activated with europium (Eu).Both were subjected to a pre-treatment comprising ball-milling therelatively large phosphors (5-6 um) until a crystal size of 1 um orsmaller was reached. The phosphors were then treated with polyfunctionalpolymers, including carboxylic acid, amino, and sulphonic acid groups.The slurry was alkaline stabilized to final pH 9.8-10.0. The ultimatedensity of the prepared phosphor slurries was 150 g/l. A slight degreeof (reversible) aggregation of the crystals was, if necessary, remediedby treating the slurry ultrasonically for 1 minute (energy 40W).

(b) Preparation of the phosphor-antibody conjugates

Two methods were tested

1) The covalent coupling of phosphors containing carboxyl groups amongothers, to antibodies by means of a spacer of ε-aminocapronic acidintroduced with water-soluble carbodiimide (EDC) (Molday et al., 1975;Rembaum, 1979).

This procedure was carried out as follows: The original phosphor slurrywas re-suspended in 10 ml 0.01M ε-amino capronic acid (Merck, Darmstadt,Western Germany) and the pH was adjusted to 5.0 with HCl. Density was 25mg/ml. In 15 minutes, 4×2.5 mg 1-ethyl-3-(3-dimethylaminopropyl)carboiimide (EDC) (Serva, Heidelberg, Western Germany) was added. Thereaction period was 2 hours at 4 C. Thereafter the crystals werecentrifuged (10 minutes, 1200x g), and washed 3 times with 0.15M NaCl.To 2.0 ml phosphor slurry containing 50 mg solid, 10 mg EDC was added; 1mg goat anti-mouse total immunoglobulin (Nordic, Tilburg, TheNetherlands) was dissolved in 100 ul 0.15M NaCl and slowly added inabout 50 minutes. The reaction was carried out at 4 C., with slowstirring for 2 hours. The reaction was then stopped with 0.2 ml 0.1Mglycine in H₂ O, and the crystals were washed 3 times with cold 0.15%NaCl solution (phosphate-buffered) (pH 7.2-7.4).

2) Coupling via strong ionic binding (adsorption).

The original phosphor slurries were diluted in 0.9% NaCl-0.15M HEPESbuffer (9:1; pH 7.4) to a density of 0.5 g/l. With careful stirring,goat anti-mouse total immunoglobulins (Nordic, Tilburg, The Netherlands)dissolved in the same buffer were added to 1 ml crystal slurry, finalconcentrations ranging from 0.25-0.005 mg/ml. Crystal aggregates werere-suspended by an ultrasonic treatment (at 4 C.), for 1 minute, 40Wenergy.

(c) Immunocytochemical reactions with phosphor-antibody conjugates

The phosphor conjugates prepared by the methods described above weretested for Ficoll-isopaque isolated human mononuclear cells marked witha mouse Leu 3a monoclonal antibody against T helper/inducer cells(Becton Dickinson, Mountain View, Calif.). To 1 ml cell slurry in 0.9%NaCl-0.15M HEPES (9:1; pH 7.4) containing 1 million of cells, 150 ul Leu3a (1:100) was added. The cells were incubated at room temperature for30 minutes and washed 3 times with medium. Subsequently, to 1 ml cellsuspension, 5-10 ul phosphor-antibody conjugate was added (density0.5%). The reaction period was 15 minutes at room temperature. The cellswere then centrifuged at 150 g for 10 minutes, carefully re-suspended,and washed with medium. There was also prepared a BSA-phosphor conjugatefor control purposes.

(d) Fluorescence-microscopy of phosphors and phosphor-antibody-labelledcells

The phosphors and phosphor-labelled cells were inspected with afluorescence microscope (Zeiss, Oberkochen, Western Germany), fittedwith a HBO 50W Mercury lamp and a filter set for excitation with UVlight (2 mm UG 1-TK 400). An LP 420 filter served as an emission-filterfor the blue phosphor crystals and an LP 590 filter for the red phosphorcrystals (Schott, Mainz, Western Germany). The objective used was aZeiss Neofluar 63 X, N.A. 1.25 phase-contrast lens. Total magnificationwas 630X. The phosphors were also inspected with a scanning electronmicroscope with a fluorescence-illuminator built-in in the vacuumchamber (Leitz, Wetzlar, Western German) (Ploem and Thaer, 1980). Thissystem makes it possible to view the cathode luminescence of thephosphors generated by the electron beam with the high-aperturefluorescence optics. Emission filters and objective were the same asdescribed above.

(e) Luminescence assays of phosphor crystals by means of microscopephotometry and flow cytometry

To study the slow luminescence phenomena of the phosphors in the time,the fluorescence microscope was fitted with a 1000W Xenon flash lamp(duration of pulse about 6 microseconds). The luminescence signals weremeasured with a Zeiss SF microscope-photomultiplier and displayed on aHewlett Packard 1744A memory oscilloscope. The measuring microscope wasinterfaced to a PDP 11 microcomputer (Digital Equipment) for automaticcontrol of the Xenon flash lamp and data handling. A program was writtenfor "time-resolved" luminescence measurements of immunophosphor labelledlymphocytes.

Flow cytometry of phosphor crystal slurries was conducted with a FACS IVcell sorter (Becton Dickinson, Mountain View, Calif.), fitted with anargon ions laser tuned to 100 mW UV (350-360 nm). Luminescence wasmeasured using an LP 420 and an LP 590 emission filter for the flue andred phosphor crystals, respectively. The second flow cytometer used wasan ICP 22 (Ortho Diagnostics) with an HBO 100 lamp as the excitationlight source and fitted with a filter set for UV excitation. Theemission filters were the same as described for the FACS IV experiments.

(f) Results

The two different methods of preparing phosphor-antibody conjugates werecompared. The covalent coupling of phosphor crystals to immunoglobulins(method 1) led to strong irreversible aggregation of the crystals thatcould not be remedied with ultrasonic treatment. Consequently, the yieldof unreacted antibody-coupled crystals was very low. As a result theimmunocytochemical results of the experiments with Leu-3a-markedlymphocytes were not optimal. Phosphor-marked lymphocytes were observed,it is true, but the major part of the cells turned out to be in largeconglomerates of cells and crystals.

Method 2, the adsorption of protein onto phosphor crystals was found notonly to be simpler to perform, but additionally led to significantlybetter results. Specific colouration of the T helper/inducer cell withphosphor-antibody conjugates was observed. The immunological controlsused, i.e. mononuclear cells not treated with Leu 3a gave no appreciablereaction with the goat anti-mouse Ig coupled phosphor. Incubations ofuncharged phosphor crystals with cells gave no bonding. Furthermore, aBSA phosphor conjugate also prepared for control purposes did not resultin significant colouration with mononuclear cells (whether or not markedwith Leu 3a).

In all fluorescence microscopy studies, phase-contrast microscopy wasused to identify and exclude aspecifically labelled monocytes.

Time-resolved luminescence measurements of immunophosphor labelled cellsshowed at least a 1 decade improved signal to noise ratio (luminescencecontrast) in comparison to conventional cytofluorometry.

The luminescence intensity of the crystals, generated by a Xenon flashlamp was large enough for it to be measured with a Zeiss Photometermicroscope and to record the luminescence decay in the time via a memoryoscilloscope. The half-life of the luminescence was in the order ofmagnitude of milliseconds.

It was possible to detect the luminescence of the individual phosphorcrystals with flow cytometry (both with laser excitation on the FACS IV,and HBO 100 excitation on the ICP 22).

REFERENCES

1) Pratt J. J., Woldring M. G., Villerius L.: Chemiluminescence-linkedimmunoassay. J Immunol Meth 21:129:184, 1978

2) Simpson J. S. A., Campbell A. K., Ryall M. E. T., Woodhead J.: Astable chemiluminescent-labelled antibody for immunological assays.Nature 279: 646-647, 1979

3) Hastings J. W., Wilson T.: Bioluminescence and chemiluminescence.Photochem Photobiol 23: 461-473, 1976

4) Oi V. T., Glaser A. N., Stryer L.: Fluorescent phycobiliproteinconjugates for analysis of cells of molecules. J Cell Biol 93: 981, 1982

5) Molday R. S., Dreyer W. J., Rembaum A., Yen S. P. S.: New immunolatexspheres: visual markers of antigens on lymphocytes for scanning electronmicroscopy. J Cell Biol 64: 75-88, 1975

6) Jongkind J. F., Verkerk A., Visser W. J., van Dongen J. M.: Isolationof autofluorescent "aged" human fibroblasts by flow sorting. Exp CellRes 138: 409-417, 1982

7) Soini E. and Kojola H.: Time-resolved fluorometer for lanthanidechelates-A new generation of nonisotopic immunoassays. Clin Chem 29: 65:68, 1983

8) Hemmila, Dakubu S., Mukkala V. M., Siitari H., Lowgren T.: EuropiumAs a label in time resolved immunofluorometric assays Anal Biochem 137:335-343, 1984

9) Rembaum A.: Microspheres as immunoreagents for cell identification.In: Flow cytometry and sorting. Eds. Melamed M. R., Mullaney P. F.,Mendelsohn M. L., p 335-347, 1979

10) Ploem J. S., Thaer J. S.: Luminescence studies with an integratedinstrument permitting SEM and fluorescence microscopy of the samespecimen. Proc Roy Microsc Soc 15: 9-10, 1980

11) Geuze H. J., Slot J. W., van der Ley P. A., Scheffer R. C. T.: Useof colloidal gold particles in double labelling immunoelectronmicroscopy of ultrathin frozen tissue section. J Cell Biol 89: 653, 1981

12) Amante L. et al: Conjugation of immunoglobulins with tetramethylrhodamine isothiocyanate. Comparison between the amorphous and thecrystalline fluorochrome. J Immunol Methods 1(3): 289-301, 1972

13) European Patent Application 0002963 Eastman Kodak Company (July1979): Aqueous stabilized fluorescent labels, proteins labelledtherewith and methods of use.

14) U.S. Pat. No. 4,000,252. K. Kosak (December 1976):Immunoscintillation cell.

We claim:
 1. An aqueous stabilized suspension of inorganic crystallinephosphor particles having surfaces provided with charged groups and aparticle size of 5 um or less, said inorganic crystalline phosphorparticles carrying a macromolecular biological substance selected fromthe group consisting of immunoglobulins, lipoproteins andpolynucleotides, said macromolecular biological substance being boundeither covalently or by physical adsorption to said inorganiccrystalline phosphor particles which can function as a luminescent labelof said macromolecular biological substance.
 2. An aqueous stabilizedsuspension as claimed in claim 1 in which the phosphor label has aparticle size of 1 um or less.
 3. An aqueous stabilized suspension asclaimed in claim 1 in which the phosphor has a particle size of 0.02 umto 5 um.
 4. A process for preparing an aqueous stabilized suspension ofinorganic crystalline phosphor particles having surfaces provided withcharged groups and a particle size of 5 um or less said inorganiccrystalline phosphor particles carrying a macromolecular biologicalsubstance selected from the group consisting of immunoglobulins,lipoproteins and polynucleotides comprising the step of binding saidmacromolecular biological substance either covalently or by physicaladsorption to said inorganic crystalline phosphor particles which canfunction as a luminescent label of said macromolecular biologicalsubstance.
 5. A process according to claim 4 in which the phosphor labelhas a particle size of 1 um or less.
 6. A process according to claim 4in which the phosphor label has a particles size of 0.02 to 5 um.
 7. Aprocess as claimed in claim 4 in which the macromolecular substance isbonded to a crystalline phosphor by physical adsorption.
 8. A processaccording to claim 7 in which the bonding is realized at a pH in thevicinity of the isoelectric point of the macromolecular substance.
 9. Aprocess as claimed in claim 4 in which the macromolecular substance iscovalently bonded to the crystalline phosphors via spacer molecules. 10.A process as claimed in claim 9 in which the bonding is realized via aspacer of E-aminocapronic acid introduced by means of water-solublecarbodiimide.
 11. A method for the immunological or immunocytochemicaldetection of a chemical substance which can be bound specifically byantibodies, said method comprising the steps of (1) contacting a samplepossibly containing said substance with an aqueous stabilized suspensionof inorganic crystalline phosphor particles having surfaces providedwith charged groups and a particle size of 5 um or less, said inorganiccrystalline phosphor particles carrying specific antibodies bound eithercovalently or by physical adsorption to said crystalline phosphorparticles which function as a luminescent label of said antibodies, and(2) detecting the luminescent label which is indicative of the presenceof said antibodies bound to said substance.
 12. The method as claimed inclaim 11 wherein the detecting is accomplished by fluorescencemicroscopy or flow cytometry with UV excitation or by scanning electronmicroscopy with electron excitation.
 13. The method as claimed in claim11 wherein the detecting is accomplished by time-resolved luminescenceassays.
 14. A method for the simultaneous detection of at least twodifferent antigens in a biological sample, comprising the steps of (1)treating said sample either simultaneously or consecutively with a firstaqueous stabilized suspension of first inorganic crystalline phosphorparticles having surfaces provided with charged groups and a particlesize of 5 um or less, said inorganic crystalline phosphor particlescarrying a first specific antibody capable of binding specifically toone of the antigens to be detected and being bound either covalently orby physical adsorption to said first inorganic crystalline phosphorparticles which function as a luminescent label of said first specificantibody and with at least one second aqueous stabilized suspension ofsecond inorganic crystalline phosphor particles having surfaces providedwith charged groups and a particle size of 5 um or less, said inorganiccrystalline phosphor particles carrying a second specific antibodycapable of binding specifically to another of the antigens to bedetected and being bound either covalently or by physical adsorption tosaid second inorganic crystalline phosphor particles which function as aluminescent label of said second specific antibody, and (2) detectingthe respective luminescent labels which can be distinguished on thebasis of their mutually different spectral characteristics orluminescence decay times and are each indicative of the presence ofantibody bound to one of the antigens to be detected.